title: Modulation of chondrocyte proliferation by ascorbic acid and BMP-2.
abstract: Chondrocytes show an unusual ability to thrive under serum-free conditions as long as insulin, thyroxine, and cysteine are present. Studies with sternal chondrocytes from chick embryos indicate that thymidine incorporation in chondrocytes cultured under serum-free conditions is 30-50% of that seen with fetal bovine serum (FBS). In contrast, skin fibroblast proliferation in serum-free culture is <5% of that seen with serum. Addition of 30-50 microM ascorbic acid to serum-free medium stimulates chondrocyte proliferation 4-5x, resulting in levels of thymidine incorporation higher than that seen with 10% serum. Three to five hours of ascorbate exposure is sufficient to stimulate proliferation, with maximal stimulation seen after 12-15 h. Bromo-deoxyuridine (BrdU) labelling indicated that approximately 25% of chondrocytes transit S phase during a 4-h period (16-20 h after ascorbate). Once maximal stimulation is reached, the proliferation rate remains fairly constant over at least 40 h. Ascorbate therefore increases the steady-state level of chondrocytes in the cycle. Because the stimulation of chondrocyte proliferation was greater than the net increase in cell numbers, we examined the level of apoptosis. Nuclear morphology, terminal uridine nucleotide end-labelling (TUNEL) assay, and 7-AAD/Hoechst dye FACS analyses all indicated that approximately 15% of the ascorbate-treated chondrocytes were undergoing apoptosis, while only 5% of the control chondrocytes were apoptotic. When prehypertrophic chondrocytes from the cephalic region of embryonic sternae were stimulated to undergo hypertrophy with rhBMP-2 + ascorbate, levels of apoptosis were similar to that seen with ascorbate alone. In contrast, treatment of caudal chondrocytes with BMP plus ascorbate does not induce hypertrophy, and the proportion of apoptotic cells was less than that seen with ascorbate alone. These results imply that in chondrocytes the transition to hypertrophy is associated with a decreased number of proliferating cells and a relatively high level of apoptosis.
authors.affiliation: Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003, USA.
authors.forename: R
authors.initials: R
authors.lastname: Venezian
authors.forename: B J
authors.initials: BJ
authors.lastname: Shenker
authors.forename: S
authors.initials: S
authors.lastname: Datar
authors.forename: P S
authors.initials: PS
authors.lastname: Leboy
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001205:Ascorbic Acid / Q000494:pharmacology*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002452:Cell Count / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects* / Q000473:pathology; D006984:Hypertrophy; D013936:Thymidine / Q000378:metabolism; D016212:Transforming Growth Factor beta; D014316:Tritium
chemical_list: D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D016212:Transforming Growth Factor beta; D014316:Tritium; D001205:Ascorbic Acid; D013936:Thymidine